Process for alcoholic fermentation of lignocellulosic biomass

ABSTRACT

A process for the production of ethanol wherein a hydrolyzed lignocellulosic biomass is fermented in the presence of a stillage residue. The fermentation of cellulosic hydrolysates is improved by adding prior to and/or during fermentation a stillage residue side stream from a corn starch-to-ethanol process as a nutrient source for the yeast organisms used in the fermentation. Stillage residues from the grain dry mill ethanol producing process, including the whole stillage, wet cake, thin stillage, and/or syrup are added to assist as a nitrogen and nutrient source for the fermentive processes. The stillage residue is produced by any grain-to-ethanol process.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 12/644,935, filed Dec.22, 2009, which claims the benefit of priority from U.S. ProvisionalPatent Application No. 61/140,451 filed Dec. 23, 2008, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to the production of ethanolfrom biomass and is particular to an improved process for alcoholicfermentation of lignocellulosic biomass.

BACKGROUND OF THE INVENTION

World energy consumption is predicted to increase 54% between 2001 and2025. Considerable effort is being directed towards the development ofsustainable and carbon neutral energy sources to meet future needs.

Biofuels are an attractive alternative to current petroleum-based fuels,as they can be utilized as transportation fuels with little change tocurrent technologies and have significant potential to improvesustainability and reduce greenhouse gas emissions.

Ethanol is a liquid alcohol made up of oxygen, hydrogen and carbon andis obtained by the fermentation of sugar or converted starch containedin corn grains or converted cellulose from other agricultural oragri-forest feedstocks. The fermentation broth is distilled anddehydrated to create a high-octane, water-free alcohol. Ethanol isblended with gasoline to produce a fuel which has environmentaladvantages when compared to gasoline, and can be used ingasoline-powered vehicles manufactured since the 1980's. Mostgasoline-powered vehicles can run on a blend consisting of gasoline andup to 10% ethanol, known as “E-10”.

In North America the feedstock is primarily corn grain, while in Brazilsugar cane is used. However, there are disadvantages to using potentialfood or feed plants to produce ethanol and the availability of suchfeedstock is limited by the overall available area of suitableagricultural land.

The term cellulosic ethanol, describes ethanol that is manufactured fromlignocellulosic biomass. There are many different sources oflignocellulosic biomass. The sources may be grouped into four maincategories: (1) wood residues (including sawmill and paper millrejects), (2) municipal paper waste, (3) agricultural residues(including corn stover, corn cobs and sugarcane bagasse), and (4)dedicated energy crops (mostly composed of fast growing tall, woodygrasses such as switch grass and Miscanthus).

Lignocellulosic biomass is composed of three primary polymers that makeup plant cell walls: Cellulose, hemicellulose, and lignin. Cellulosefibers are locked into a rigid structure of hemicellulose and lignin.Lignin and hemicelluloses form chemically linked complexes that bindwater soluble hemicelluloses into a three dimensional array, cementedtogether by lignin. The complexes cover cellulose microfibrils andprotect them from enzymatic and chemical degradation. These polymersprovide plant cell walls with strength and resistance to degradation.This makes them a challenge to use as substrates for biofuel production.

Production of ethanol from cellulose via fermentation is a complexprocess that starts with feed preparation and is followed by biochemicalconversion and distillation.

Delivery of biomass starts with selective harvesting, transportation,storing and reducing steps. Biochemical conversion of lignocellulosicbiomass to ethanol involves four steps: (1) High pressure treatment ofraw lignocellulosic biomass to make the complex polymers more accessibleto enzymatic breakdown; (2) production and application of special enzymepreparations (cellulases and hemicellulases) that hydrolyze pretreatedplant cell-wall polysaccharides to a mixture of simple sugars; (3)fermentation, mediated by bacteria or yeast, to convert these sugars toethanol; and (4) ethanol distillation and dehydration.

One variable in the composition of biomass that affects the conversionto energy is lignin. There is evidence that lignin inhibits the processof breaking down biomass to sugars for fermentation. Lignin and somesoluble lignin derivatives inhibit enzymatic hydrolysis and fermentationprocesses. Thus, it is desirable to use a lignocellulosic feedstockwhich is low in lignin. The lignin content of corncobs, (less than 8% byweight) is low, which would make this a good biomass feedstock for theproduction of ethanol. However the hemicellulose content of corncobs ishigh, about 30 to 40% of the total dry matter. Moreover, much of thehemicellulose is acetylated which means that breakdown and liquefactionof the hemicellulose leads to the formation of acetic acid. This is aproblem, since the acid is a powerful inhibitor of the ethanolfermentation process. It remains in the pretreated biomass and carriesthrough to the hydrolysis and fermentation steps.

Diverse techniques have been explored and described for the pretreatmentof size-reduced biomass with the aim of producing a substrate that canbe more rapidly and efficiently hydrolyzed to yield mixtures offermentable sugars.

These approaches have in common the use of conditions and procedureswhich greatly increase the surface area to which aqueous reactants andenzymes have access. In particular, increasing the percentage of thecellulosic materials that are opened up to attack decreases the timeneeded to hydrolyze the cellulose polymers to simple sugars. However,pretreatments of lignocellulosic biomass, such as steam explosion, mayresult in extensive cellulose breakdown and, to a certain extent, to thedegradation of hemicellulose. This results in the production of aceticacid and furfural. Some pretreatment methods employ hydrolytictechniques using mineral acids (hemicellulose hydrolysis) and alkalis(lignin removal).

Pretreatments involving mineral acids (including SO₂) primarilysolubilize the hemicellulose component of the feedstock while the use oforganic solvents and alkalis tends to co-solubilize lignin andhemicellulose.

The resulting product streams (called pre-hydrolysates) are usuallyseparated thereafter into liquid and solid (cellulose) phases. If noseparation or detoxification is included in the process, a complexmixture of toxic compounds such as acetic acid and furans will becarried forward to the hydrolysis and fermentation steps. The inhibitorycompounds significantly reduce enzyme performance, biocatalyst growth,rates of sugar metabolism, and final ethanol titer due to incompleteconversion of the glucose to ethanol.

The mentioned inhibitors are generally removed through a dedicated stepto detoxify pretreatment hydrolysates before fermentation.Detoxification requires additional equipment, e.g. solid-liquidseparation, storage tanks, and may also require the addition ofchemicals such as calcium hydroxide for over liming, acid forneutralization before fermentation and high yeast nutrient loads, henceadded process complexity.

This additional process complexity results in increased capitalequipment and operating costs. Therefore, it would be desirable to avoidthe need to detoxify completely biomass prehydrolysate prior to theenzymatic hydrolysis and fermentation steps.

Fermentation of sugars by yeast (e.g. Saccharomyces cerevisiae) is themost common method for converting sugars released from biomassfeedstocks into fuels, such as ethanol. Yeasts are living organisms,unicellular fungi that need carbon, nitrogen, vitamins, and minerals forgrowth and reproduction. If compared to corn grain mash, lignocellulosichydrolysates are not nutritionally balanced for yeast and most need tobe fortified with additional macronutrients like nitrogen.

Nitrogen is an essential element needed to avoid sluggish and stuckfermentation. Nitrogen deficiency will cause problems in fourfundamental ways. The first three are related to each other as follows:(1) protein synthesis is limited; (2) cell count is limited because theproteins are the bricks used to built new cells, and (3) fermentationkinetics are slowed down due to the reduced cell count. The fourthmanner in which nitrogen deficiencies can cause sluggish fermentation isthrough a decrease in the efficiency of the sugar transporters in theyeast cell membrane, causing a significant decrease in fermentationkinetics at the cellulose level.

Yeast accessible nitrogen is composed of two portions, organic orassimilable nitrogen and inorganic nitrogen (ammonia). Advantageousfermentation broths contain a balance of yeast available nitrogen fromboth assimilable amino nitrogen and inorganic nitrogen. Therefore, thefermentation step typically requires external nutrient supplementation.

Another major barrier in the efficient use of biomass-derived sugars isthe lack of industrial grade yeast that can grow and function optimallyin challenging, stressful environments created by lignocellulosicbiomass pretreatments as discussed above.

During the fermentation of a detoxified biomass hydrolysate, asignificant fraction of available sugar may be diverted by the yeastaway from ethanol production to glycerol and succinic acid production.Glycerol production in the yeast is linked to acid, ethanol, andtemperature induced stress conditions. The synthesis of glycerol occursin response to osmotic stress and therefore likely has an essential rolein cell viability.

Although yeasts with improved properties such as elevated ethanol andtemperature tolerances have been genetically engineered, such strainsare not yet used widely by the fuel ethanol industry.

All of the above mentioned problems contribute to the elevated capitalcost and operating cost of lignocellulosic ethanol production byreducing product yields, and increasing water volumes that must behandled as part of relatively dilute product streams.

Ethanol production from glucose or from grain or corn starch is now amature industry. Production of fuel ethanol from sugars present inlignocellulosic biomass, however, remains challenging with manyopportunities for improvement.

Thus, improving the throughput and reducing the costs associated withethanol production from lignocellulosic biomass, is critical to theestablishment of a viable industry.

SUMMARY OF THE INVENTION

It is now an object of the present invention to provide a process whichovercomes at least one of the above disadvantages.

The inventors of the present application have realized that processintegration of cellulosic ethanol production with residue streams froman existing starch-based process would reduce both capital and operatingcosts, which remain high by comparison with those of corn.

The inventors have further discovered that savings in capital andoperating costs can be realized by developing improved cellulose toethanol processes wherein the fermentation of cellulosic hydrolysates isimproved by adding prior to and/or during fermentation a residue sidestream or stillage residue from a corn starch-to-ethanol process as anutrient source for the yeast organisms used in the fermentation. Thisnutrient source not only improves the fermentation rate and efficiency,but also improves the resistance of the yeast organisms to acidic and/orother impurities or inhibitors in the cellulosic hydrolysates. Theseimpurities and inhibitors may have been created or added duringcellulose pretreatment and cellulose hydrolysis. Using a nutrient streamfrom a corn to ethanol process to fortify lignocellulosic hydrolysateseven provides the possibility of carrying out the fermentation processwith only partial washing, limited detoxification or pH adjustment ofthe cellulose hydrolysates, or even without any washing, detoxification,or pH-adjustment. All of this is achieved by simply adding prior and/orduring fermentation a side stream or stillage residue from a grainstarch-to-ethanol process.

In one preferred aspect, the invention provides a cellulose-to-ethanolprocess wherein fermentation of cellulosic hydrolysates derived fromligno-cellulosic biomass can be carried out with full washing anddetoxification, partial washing, detoxification or pH-adjustment of thecellulosic hydrolysates, or without any prior washing or detoxificationof the steam explosion pretreated biomass hydrolysates.

In a further preferred aspect, the invention provides acellulose-to-ethanol process wherein stillage residue streams from agrain or corn grain starch-to-ethanol dry mill process are added duringfermentation to reduce the need for pH adjustment or external nutrientsupplementation.

In one aspect, the present invention resides in a process for theproduction of ethanol, the process comprising the step of fermenting ahydrolyzed lignocellulosic biomass in the presence of a stillageresidue, the stillage residue is produced by a whole grainstarch-to-ethanol process.

In a preferred aspect, the stillage residue is selected from the groupconsisting of whole stillage, thin stillage, wetcake, syrup, and anycombination thereof.

In a preferred aspect, the process further comprises a propagation stepwhereby yeast is conditioned and grown prior to the step offermentation.

In a preferred aspect, the hydrolyzed lignocellulosic biomass isproduced by acid pre-treatment wherein the acid catalyst is a mineralacid or a carboxylic acid.

In a preferred aspect, the hydrolyzed lignocellulosic biomass isselected from the group consisting of agricultural residues, purposegrown crops, woody biomass, and any combination thereof.

In a preferred aspect, the hydrolyzed lignocellulosic biomass isobtained from corn cobs.

In a preferred aspect, the ethanol is produced by fermentation with aethanologenic organism.

In a preferred aspect, the ethanologenic organism is a prokaryoticorganism.

In a preferred aspect, the ethanologenic organism is selected from thegroup consisting of Escherichia coli, Klebsiella oxytoca, and Zymomonasmobilis, Clostridium thermocellum.

In a preferred aspect, the ethanologenic organism is a eukaryoticorganism.

In a preferred aspect, the eukaryotic organism is selected from thegroup consisting of Saccharomyces cerevisia, Pichia stipitis.

In another aspect, the present invention resides in a process comprisingthe step of propagating yeast in the presence of a stillage residue froma whole grain starch-to-ethanol process.

In a preferred aspect, the stillage residue is selected from the groupconsisting of whole stillage, thin stillage, wetcake, syrup, and anycombination thereof.

In another aspect, the present invention resides in a process for theproduction of cellulosic ethanol from lignocellulosic biomass,comprising the steps of: pretreating the lignocellulosic biomass todecompose the lignocellulosic biomass into fibrous solids; hydrolyzingthe fibrous solids with enzymes to produce cellulose sugars; andfermentating the cellulose sugars in the presence of a stillage residuefrom a whole grain starch-to-ethanol dry mill process.

In a preferred aspect, the stillage residue is selected from the groupconsisting of whole stillage, thin stillage, wetcake, syrup, and anycombination thereof.

In a preferred aspect, the step of pretreating the lignocellulosicbiomass includes process conditions including the step of exposing thelignocellulosic biomass to steam in a reaction vessel at an elevatedtemperature and reaction pressure for a preselected exposure time, andreleasing the reaction pressure for explosive decomposition of thelignocellulosic bioimass.

In a preferred aspect, the elevated temperature is in the range of190-210° Celsius, the reaction pressure is between 190 to 275 psig, andthe preselected exposure time is between 3 to 10 minutes.

In a preferred aspect, the elevated temperature is 205 degrees Celsius,the reaction pressure is 235 psig, and the preselected exposure time is8 minutes.

In a preferred aspect, the pressure is released within less than 1000milliseconds.

In a preferred aspect, the pressure is released within 300 milliseconds.

In a preferred aspect, the process conditions are selected for theachievement of a severity index of 3.9 to 4.1.

In a preferred aspect, the severity index is 4.0.

In a preferred aspect, the enzymatic hydrolysis is carried out at 10-30%consistency, and at a temperature of 40-60° Celsius and a pH 4.5 to 5.5.

In a preferred aspect, the fermentation step is carried out at 10-30%consistency, 30-37° C. and a pH of 5.2 to 5.9.

In a preferred aspect, the enzymatic hydrolysis of solids generatedduring the pressure release steps is carried out at a temperature of 50°C., pH 5.0 until completion.

In a preferred aspect, the fermentation step is carried out at atemperature of 35° C., and at a pH of 5.3 until completion.

In a preferred aspect, the process includes a process arrangement stepfor step of collecting and processing fermentation products fordistilling fuel grade ethanol.

In a preferred aspect, the process arrangement step for distilling fuelgrade ethanol includes a distillation portion, a condensation anddehydration portion, a separation and drying portion and an evaporationportion.

In a preferred aspect, the process arrangement produces hot ethanolvapor and thin stillage.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent upona reading of the detailed description and upon reference to the drawingsin which:

FIG. 1 shows the effect of pH on the fermentability of unwashed,undetoxified corncob hydrolysate from steam explosion pretreatment inthe absence of external nutrient supplementation;

FIG. 2 shows the effect of commercial yeast nutrient load on thefermentability of unwashed, undetoxified corncob hydrolysate from steamexplosion pretreatment, when the fermentation is carried out at a lowload of pH adjustment chemical (pH 5.3);

FIG. 3 shows the effect of yeast commercial nutrient load on thefermentability of unwashed, undetoxified corncob hydrolysate from steamexplosion pretreatment, when the fermentation is carried out at a highload of pH adjustment chemical (pH 5.9);

FIG. 4 shows the effect of nutrient sources on the fermentability ofunwashed, undetoxified corncob hydrolysate from steam gun pretreatmentwhen the fermentation are carried out at a low load of pH adjustmentchemical (pH 5.3).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before explaining the present invention in detail, it is to beunderstood that the invention is not limited to the preferredembodiments contain herein. The invention is capable of otherembodiments and of being practiced or carried out in a variety of ways.It is to be understood that the phraseology and terminology employedherein are for the purpose of description and not of limitation.

The abbreviations used in figures and tables have the following meaning:

-   h, hours-   g/l, gram per liter-   ml, milliliter-   DM, Dry matter-   t90%, time (hours) to reach 90% of the maximum theoretical    conversion of glucose to ethanol

Table 1 shows the impact of pH on fermentation rates;

Tables 2 A and 2B show the effect of pH adjustment on the cost of pHadjustment chemical (aqueous ammonia) used in unwashed, undetoxifiedcorncob prehydrolysates hydrolysis and fermentation;

Table 3 shows the impact of commercial yeast nutrient load on glucose toethanol rates;

Tables 4 A and 4B show composition analysis of alternative yeastnutrients; and

Table 5 shows the impact of nutrient sources and loads on fermentationrates of unwashed, undetoxified corncob hydrolysates when thefermentation is carried out at a low load of pH adjustment chemical (pH5.3).

The invention is directed to ethanol from biomass processes andespecially to cellulose fermentation processes. In particular, theinvention is directed to processes intended to achieve fermentation ofcellulosic hydrolysates which include one or more acidic or otherimpurities or inhibitors of the yeast used in the fermentation step.Preferably, the invention provides a process which partially or fullyobviates the step of washing or detoxification of a lignocellulosichydrolysate prior to fermentation.

In a common dry mill grain or corn grain ethanol producing plant, theethanol is removed from the fermented mash in a distillation column.After the ethanol is removed, the remaining residue is removed asstillage residue. The stillage residue which is not refined is known aswhole stillage. The whole stillage can be run through a solid-liquidseparation step producing a solid stream of residue, also known as wetcake, and a liquid stream of residue, also referred to as thin stillage.The thin stillage can be further processed to increase the solidsconcentration by evaporation resulting in Condensed Distillers Solublesor Syrup. Typically the Syrup is mixed back with the separated solidstream or wet cake and fed to a rotary drum dryer to remove theremaining moisture. The resulting dry solids are typically referred toas Dried Distillers Grains and Solubles or “DDGS”, and sold as animalfeed. However, the inventors have discovered that the stillage residuesfrom the grain dry mill ethanol producing process, including the wholestillage, wet cake, thin stillage, and/or syrup present a low costprotein and nitrogen nutrient source for fermentive processes.

Adding the stillage residue as a nutrient source in accordance with theinvention can reduce the amount of pH adjustment chemical required andreduce or eliminate the need for expensive nutrient supplements.

This process of adding the stillage residue as a nutrient source can beused with any lignocellulose to ethanol producing process includingthose using corncob or other lingo-cellulosic material as the startingcellulosic material. In particular, this process also applies to ethanolproducing processes including steps of cellulose pretreatment, andhydrolysis methods.

Hemicellulose is a heteropolymer or matrix polysaccharide which ispresent in almost all plant cell walls along with cellulose. Whilecellulose is crystalline, strong, and resistant to hydrolysis,hemicellulose has a random, amorphous structure with little strength.Hydrolysis of hemicellulose can be relatively easily achieved with acidsor enzymes. Hemicellulose contains many different sugar monomers. Forinstance, besides glucose, hemicellulose can include xylose, mannose,galactose, rhamnose, and arabinose. Xylose is the monomer present in thehighest amount.

While cellulose is highly desirable as a starting material forbiochemical ethanol production, hemicellulose and most of its hydrolyticdegradation products interfere with the downstream fermentation ofglucose from cellulose. In particular, xylose derivatives anddegradation products, and acetic acid, all of which are products ofhemicellulose hydrolysis, are strong inhibitors of glucose fermentation.

EXAMPLE 1

Complete enzymatic digestion of corncob pre-hydrolysates was carried outwith a commercial enzyme product GC220 (Genencor) at 3.0% load (w/w,DM), 50° C., and pH 5.0.

The preferred enzymatic digestion conditions were found to be 10-30%consistency of the prehydrolysate, a temperature of 40-60° C. and a pHof 4.5-5.5.

Fermentations were carried out using Ethanol Red™, a commercialindustrial grade C6-fermenting yeast from Fermentis (division ofLesaffre group), as a benchmark yeast. Yeast inoculation was carried outby adding 6.67 g dry yeast per kilogram of corncob hydrolysates leadingto an average yeast population of 10⁸ cells/ml hydrolysate afterrehydration.

The benchmark conditions for the fermentation experiments were 35° C.,pH 5.9, using commercial nutrient (Goferm™, 8.3 g/kg corncobhydrolysates or 4.0% (w/w, DM) load). Preferred fermentation conditionswere found to be a consistency of 10-30%, a temperature of 30-37° C. anda pH of 5.2 to 5.9.

Fermentations were carried out in 1-liter beakers. The key parametersused were fermentation rates and yield. Fermentation rates and yield ofbatch or continuous steam explosion pretreated corncob were assessedwith respect to pH adjustment chemical usage and yeast nutrient needs.

pH adjustment of unwashed, undetoxified hydrolyzed corncobsprehydrolysate was carried out prior to fermentation using differentquantities of liquid ammonia (30%, w/w). Starting pH's ranged from 5.0to 5.9.

Yeast nutrient needs were first evaluated using different loads of acommercial yeast nutrient (Goferm) with respect to fermentation ratesand yield. The performance of Goferm was used as a benchmark for thescreening and identification of industrial side streams or stillageresiduess.

The industrial side streams or stillage residues evaluated were wholeand thin stillage, wet cake and syrup from a corn starch-to-ethanol drymill as well as heavy steep water from a wet mill.

Glucose, xylose, ethanol, glycerol and carboxylic acid concentrationswere determined by HPLC analysis. Quantification of soluble productsfrom pretreatment, enzymatic hydrolysis and fermentation were carriedout by HPLC analysis. Target molecules were monitored to determine therelative contents of cellulose and downstream inhibitors in theprehydrolysate obtained. The target molecules were sugar monomers suchas glucose and xylose as well as toxic compounds such as differentcarboxylic acids, namely acetic acid, succinic acid and lactic acid anddegradation products of carbohydrates such as glycerol, HMF and furfuralas well as ethanol.

Composition analyses of commercial yeast nutrient and industrial sidestreams or stillage residuess were performed by an external laboratory(DairyOne). The overall hydrolysis and fermentation time of thebatch-pretreated corncobs was generally less than 100 hours in total.

The summary results of the test fermentation series are plotted in FIGS.1 to 4 and Tables 1 to 5.

As shown in FIG. 1, fermentation of unwashed, undetoxified corncobpre-hydrolysates can be accomplished at reasonable ethanolconcentrations in reasonable time at pH values higher than pH 5.6 inabsence of nutrient. Glucose from corncob hydrolysates can be convertedto ethanol with a yield of 92% of the theoretical maximum, using acommercial industrial grade C6-fermenting yeast. An ethanolconcentration of 5.4% (w/v) can be reached in 23 hours to 40 hours.

Table 1 shows that lower pH in fermentation of corncob is associatedwith a slowdown of fermentation even in the presence of a high load ofcommercial yeast nutrients. The time to reach 90% of the maximumtheoretical glucose to ethanol conversion increased from 15 hours to 24hours when the pH was reduced from 5.9 to 5.2.

FIG. 2 shows that the fermentation of hydrolysates had to be carried outwith a minimum of 0.26% of commercial yeast nutrient, Goferm at pH of5.3. Tables 2A and 2B show that the minimum ammonia usage for initial pHadjustment (pH 5.3) of corncob hydrolysates prior to fermentation was 10ml ammonia (30%, w/w) per kilogram of corncob dry matter. Thiscorresponds to 2.7 g of pure ammonia per metric ton corncob dry matter.The cost of the minimum usage of ammonia was 3.4 cents per liter ofanhydrous alcohol. Starting fermentation at pH 5.9 leads to an increaseof 0.5 cents per liter of anhydrous alcohol, compared to a fermentationstarting at pH 5.3.

Table 3 shows that fermentation time increases with a decrease incommercial yeast nutrient load (source of yeast assimilable nitrogenfrom protein). Yeast growth and viability requires organic and inorganicnitrogen sources, as discussed above.

Table 4 shows that alternative yeast nutrients from a corn ethanol plantcan replace commercial yeast nutrients. The main nitrogen source incommercially available yeast nutrients is protein. The percentage ofprotein in Goferm is 51.4% (w/w, DM). FIG. 3 shows that the fermentationof pretreated corncobs can be performed with no nutrient addition if thepH is raised to 5.9 with the use of high load of preferred pH adjustmentchemical. These results were anticipated since ammonia usage to reachhigher pH prior fermentation was significantly greater and ammonia isalso widely used as inorganic nitrogen source for yeast growth.

FIG. 4 shows that side streams or stillage residuess from a starch toethanol facility can be successfully used as alternative source of yeastnutrient and allow operation of the fermentation process at pH 5.3, andat the lowest possible input of pH adjustment chemical.

Table 5 shows that fermentation rates and yields obtained with theaddition of wet cake, thin stillage and syrup as nutrients, were similarto those obtained with the yeast commercial yeast nutrient. The use ofsyrup as yeast nutrient for the fermentation of pretreated corncobhydrolysate is recommended since syrup is the final by-product of thestarch to ethanol process. Moreover, only a part of the syrup producedis normally sold at low cost ($20 per/MT as is). Disposal of theremaining syrup is a significant process issue.

The stillage residues have, among other things, protein, triglycerides,free fatty acids, vitamins, sterols, etc. that help the yeast ferment.While soluble protein is a more favorable nutrient for yeast, the wholestillage contains both soluble and insoluble protein. The thin stillageand subsequent syrup contain a greater concentration of soluble proteinthan the whole stillage. As can be seen in Table 5, the protein load ofthe wet cake is 50% more than that of the stillage and the syrup, andthe addition of wet cake as a nutrient source achieves 90% fermentationin the same amount of time as the when the stillage or the syrup areused as a nutrient source. It can also be seen from Table 5 that thewhole stillage has equal protein to that of the Goferm load 2; howeverwhen the whole stillage was used as a nutrient source, it took longer toreach 90% fermentation than when the Goferm load 2 was used as anutrient source. This is likely due to the high level of insolubleprotein in the whole stillage as compared to that of the expensiveGoferm. The Heavy Steep Water is a special case as it had a slightlyhigher load of soluble protein over the syrup but gave the worst result.This is likely due to the very high content of lactic acid 10% w/v andacetic acid 0.4% w/v as seen in Table 4b.

EXAMPLE 2

Batch steam explosion corncob pretreatment was carried out in a steamgun treatment process and experimental cellulose pretreatment setup asdescribed in U.S. Provisional Patent Application No.61/097,692—Cellulose pretreatment process.

Batch loads of 6 kg DM of 0.5 to 1 cm corncob were used per steamexplosion shot. Pressurized saturated steam at temperatures of 190 to210 degrees C. was fed into the steam gun until the desired cookingpressure was reached. Cooking pressures of 235 psig were used. After aresidence time of 8 minutes, the pressure in the steam gun was quicklyreleased. Complete pressure relief was achieved in 600 to 1000 ms.During the residence time and prior to pressure release, condensate andcooking liquids collected at the bottom of the steam gun were purgedthrough purge discharge control. Solids and gaseous reaction productsejected from the steam gun on pressure release were separated in acyclone separator. The solids collected at the bottom of cycloneseparator were subjected to lab scale hydrolysis and fermentation.

Carbohydrate composition analysis of corncobs as fed and corncobprehydrolysates collected at the bottom of the cyclone separator wascarried out at Paprican's analytical laboratory (Montreal, Qc).

Complete enzymatic digestion of corncob pre-hydrolysates was carried outwith a commercial enzyme product GC220 (Genencor) at a mediumconsistency of the corncob prehydrolysate (25%), 3.0% load of enzyme(w/w, DM), 50° C., and pH 5.0 in a 1-Liter stirred reaction vessel (150rpm). pH was adjusted with aqua ammonia (15%, w/w).

Fermentations were carried out using Ethanol Red, a commercialindustrial grade C6-fermenting yeast from Fermentis. Yeast inoculationwas carried out by adding 6.67 g dry yeast per kilogram of corncobhydrolysates leading to an average yeast population of 10⁸ cells/mlhydrolysate after rehydration.

Fermentation experiments were carried out in 1-liter beakers at 35° C.,pH 5.3, using 13.5 g as-is of syrup from a corn starch-to-ethanol drymill process. pH adjustment of unwashed, undetoxified hydrolyzed corncobprehydrolysate was carried out prior to fermentation, using differentquantities of aqueous ammonia (30%, w/w).

The key parameters used were fermentation rates and yield. Fermentationrates and yield of batch or continuous steam explosion pretreatedcorncob were assessed with respect to the usage of pH adjustmentchemical and yeast nutrient needs.

Glucose, xylose, ethanol, glycerol and carboxylic acid concentrationswere determined by HPLC analysis. Quantification of soluble productsfrom pretreatment, enzymatic hydrolysis and fermentation was carried outby HPLC analysis. Target molecules were monitored to determine therelative contents of cellulose and downstream inhibitors in theprehydrolysate obtained. The target molecules were sugar monomers suchas glucose and xylose as well as toxic compounds such as differentcarboxylic acids, namely acetic acid, succinic acid and lactic acid anddegradation products of carbohydrates such as glycerol, HMF and furfuralas well as ethanol.

TABLE 1 pH¹ 5.0 5.2 5.3 5.6 5.9 Time 90%² (h) NA³ 24 19 17 15¹Fermentation pH ²Time to reach 90% of the maximum glucose to ethanolconversion ³Not applicable (%-conversion never reached)

TABLE 2A pH 5.9 pH 5.3 (A) Volume (ml) 30% ammonia per kg DM corn cobHydrolysis 51 51 Saccharification 8 8 Fermentation 20 10 Total 79 69 (B)Cent per Liter anhydrous alcohol anhydrous alcohol Hydrolysis 2.5 2.5Saccharification 0.4 0.4 Fermentation 1.0 0.5 Total 3.9 3.4

TABLE 3 Goferm Goferm load Protein load Time 90% g-as is/kg (%, w/w, DM)(%, w/w, DM) (h) 8.3 0.78 0.4 20 5.6 0.52 0.27 23 2.8 0.26 0.13 24 1.30.13 0.07 28 0.0 0.0 0.0 49.0

TABLE 4 (A) Nutrient sources % Dry Matter % Crude Protein % Crude FatGoferm 93.7 51.4  0.6 Wetcake 32.9 36.6  8.4 Thin stillage  7.6 22.818.7 Syrup 33.7 20.9 19.8 Whole stillage 12.7 30.8 14.2 Heavy steepwater 42.3 49.2  0.3 (B) Soluble compounds concentration (g/L) Nutrientsources Glucose Xylose Lactic acid glycerol Acetic acid Wetcake 0.000.43 1.49  8.62 0.74 Thin stillage 0.10 0.59 1.96 12.69 0.36 Syrup 1.252.94 8.91 61.31 0.50 Whole stillage 0.00 0.32 5.48 12.63 0.44 Heavysteep water 42.54  44.94  96.13   3.99 4.26

TABLE 5 Nutrient sources, loads and impact on fermentation ratesNutrient load (g) Protein load Time 90% Nutrient sources as is per kg(%, w/w, DM) (h) Goferm load1 5.6 0.27 21 Goferm load2 1.3 0.07 28Wetcake 12.4 0.15 21 Thin stillage 60.0 0.10 21 Syrup 13.5 0.10 21 Wholestillage 17.0 0.07 32 Heavy steep water 5.4 0.11 40

What is claimed is:
 1. A process for the production of ethanol, theprocess comprising the step of fermenting a hydrolyzed lignocellulosicbiomass in the presence of a stillage residue, the stillage residue isproduced by a whole grain starch-to-ethanol process.
 2. The process ofclaim 1, wherein the stillage residue is selected from the groupconsisting of whole stillage, thin stillage, wetcake, syrup, and anycombination thereof.
 3. The process of claim 1, further comprising apropagation step whereby yeast is conditioned and grown prior to thestep of fermentation.
 4. The process of claim 1, wherein the hydrolyzedlignocellulosic biomass is produced by acid pre-treatment wherein theacid catalyst is a mineral acid or a carboxylic acid.
 5. The process ofclaim 1, wherein the hydrolyzed lignocellulosic biomass is selected fromthe group consisting of agricultural residues, purpose grown crops,woody biomass, and any combination thereof.
 6. The process of claim 1,wherein the hydrolyzed lignocellulosic biomass is obtained from corncobs.
 7. The process of claim 1, wherein the ethanol is produced byfermentation with a ethanologenic organism.
 8. The process of claim 7,wherein the ethanologenic organism is a prokaryotic organism.
 9. Theprocess of claim 8, wherein the ethanologenic organism is selected fromthe group consisting of Escherichia coli, Klebsiella oxytoca, andZymomonas mobilis, Clostridium thermocellum.
 10. The process of claim 7,wherein the ethanologenic organism is a eukaryotic organism.
 11. Theprocess of claim 10, wherein the eukaryotic organism is selected fromthe group consisting of Saccharomyces cerevisia, Pichia stipitis.
 12. Aprocess for the propagation of yeast, the process comprising the step ofpropagating yeast in the presence of a stillage residue from a wholegrain starch-to-ethanol process.
 13. The process of claim 12, whereinthe stillage residue is selected from the group consisting of wholestillage, thin stillage, wetcake, syrup, and any combination thereof.